UC-NRLF 


EXCHANGE 


,  BIOLOGY 

LIBRARY 

6 


The  Occurrence 

of  Aluminium,  and  its  Absorp 

tion  from  Food,  in  Dogs 


DISSERTATION 


Submitted  in  Partial  Fulfillment  of  the  Re- 
quirements for  the  Degree  of  Doctor  of 
Philosophy  in  the  Faculty  of  Pure  Science 
of  fcolumbia  University 


By 
ARNOLD  K.  BALLS 


Highland  Democrat  Print 

Peekskill,  N.Y. 

May,  1920 


The  Occurrence 

of  Aluminium,  and  its  Absorp 

tion  from  Food,  in  Dogs 


DISSERTATION 


Submitted  in  Partial  Fulfillment  of  the  Re- 
quirements for  the  Degree  of  Doctor  of 
Philosophy  in  the  Faculty  of  Pure  Science 
of  Columbia  University 


By 
ARNOLD  K.  BALLS 


Highland  Democrat  Print 

Peekskill,  N.Y. 

May,  1920 


BIOLOGY 
R 
G 


OUTLINE 

I.  Introduction. 

II.  Method. 

III.  Is  the  aluminium  of  aluminized  bread  soluble  in  the 

digestive  juices  ? 

IV.  Does  aluminium  occur  in  the  tissues  of  normal  dogs? 
V.    Is  aluminium  absorbed  from  food  containing  it  in  po- 
tentially soluble  form? 

VI.    Is  aluminium  absorbed  by  dogs  from  aluminized  bread? 


I. 

Introduction 

This  paper  describes  an  investigation  of  the  general  subject 
of  the  occurrence  of  aluminium  in  the  animal  body,  both  under 
normal  conditions,  and  on  diets  containing  aluminium. 

The  problem  is  of  considerable  theoretical  interest.  The 
occurrence  of  aluminium  in  the  bodies  of  normal  animals  has 
never  been  specially  investigated,  but  large  amounts  are  certainly 
not  present,  or  they  would  have  been  easily  recognized  ere  this. 
This  condition  is  in  marked  contrast  to  the  abundance  of  aluminium 
in  the  plant  and  mineral  worlds.  That  aluminium,  like  silicon, 
exists  in  the  animal  body  in  small  amounts,  could  even  be  assumed 
on  the  ground  of  accidental  contamination,  unless  some  specific 
eliminative  selection  takes  places,  which  would  point  biologically 
to  an  incompatibility  between  the  metal  and  the  organism.  Ab- 
sorption, and  the  retention  of  aluminium,  if  either  or  both  occur, 
bear  directly  upon  the  possibility  of  substituting  one  element  for 
another  in  the  body.  Information  concerning  such  replacements 
consequently  means  an  increase  in  our  knowledge  of  the  functions 
of  the  substances  involved. 

From  the  practical  standpoint,  the  wide  use  of  alum  baking 
powders,  especially  in  the  South,  makes  it  desirable  that  the  effect 
of  this  aluminium  should  be  determined. 

The  problem  was  first  attacked  by  Papillon1  in  1870,  who,  after 
feeding  aluminium  phosphate  to  a  young  rat,  found  that  the  bones 
contained  6.95%  of  alumina.  He  concludes  that  his  "researches 
show  it  possible  to  substitute  a  certain  quantity  of  strontium,  of 
magnesium,  or  of  aluminium  for  the  calcium  normally  occurring 
in  the  bones. 

H.  Weiske2  was  unable  to  confirm  these  results  with  aluminium, 
although  he  verified  them  with  regard  to  the  other  elements  used. 
He  recognizes  the  fact  that  such  substitutions  may  not  occur  to  a 
marked  extent  in  an  animal  kept  on  a  normal  diet;  but  that  by 

iPapillon:  Comp.  rend,  de  1'Acad.  de  sciences  (Paris),  Ixxi,  p.  372, 
(1870). 

2H.  Weiske:    Zeit.  fur  Biologie.  vlii,  239,  (1872),  ibid,  xxxi,  421,  (1895) 

V  1 


depriving  the  animal  of  a  sufficient  amount  of  the  element  to  be 
replaced,  (in  this  case  calcium)  substitution  may  be  effected. 

J.  Konig3  first  practically  applied  this  principle  of  Weiske's, 
using  food  deficient  tin  calcium  He  was,  however,  unable  to  find 
any  large  amounts  of  aluminium,  but  states  that  he  made  no  at- 
tempt to  look  for  small  amounts,  and  concedes  the  possibility  of 
their  being  present. 

It  was  in  this  unsatisfactory  condition  that  the  problem  was 
first  taken  up  at  this  laboratory. 

In  1906  House  and  Gies4  showed  that  a  concentration  of  alu- 
minium greater  than  1-65,536  moles  per  liter  markedly  inhibited  the 
growth  of  seedlings.  It  was  later  found  by  Steel5,  working  with 
Gies,  that  in  dogs,  aluminium  passed  from  the  alimentary  tract 
into  the  blood,  when  they  were  given  food  mixed  with  alum,  and 
that  when  aluminium  chloride  was  injected  intravenously  this  pro- 
cess was  reversed,  aluminium  passing  into  the  intestines  from  the 
blood. 

Kahn8,  also  working  in  this  laboratory,  showed  that  aluminium 
is  absorbed  by  dogs  from  food  prepared  with  alum  baking  powder. 

On  the  other  hand,  the  Referee  Board  of  Consulting  Scientific 
Experts  of  the  United  States  Department  of  Agriculture7  were 
unable  to  find  aluminium  in  the  blood  of  four  men  who  "took  one 
gram  of  aluminium  a  day  for  several  days."  The  form  in  which 
the  aluminium  was  taken,  however,  is  not  mentioned. 

More  recently,  Leary  and  She'ib8,  using  dogs  and  white  rats, 
which  were  fed  with  aluminium  hydroxide,  identified  aluminium  in 
the  livers  of  all  the  animals  used. 

In  our  experiments  we  have  used  dogs,  both  because  of  their 
convenience,  and  since,  of  the  animals  available,  their  metabolism 
is  nearest  to  that  of  human  beings. 

3J.  Konig-:     Zeit.  fur  Biologie..  x,  69,   (1874). 

4House  and  Gies:     Amer.  Jour.  Physiol.  xv,   (1906).     Proc.  p.  xix. 

5Steel:     Amer.  Jour.  Physiol.  xxviii,   94,   (1911). 

6Kahn:     Biochem.  Bull,   i,   235,   (1911). 

7Bull.   103,  U.  S.  Dept.  of  Agriculture. 

8Leary  and  Sheib:     Jour.  Amer.  Chem.  Soc.  xxxix,  1066,   (1917). 


II. 

Method 

Tor  description  of  the  analytical  methods  employed,  see  Balls: 
Biochemical  Bull  V,  pp.  195-202.  (1916) 

III. 

Is  the  Aluminium  of  Aluminized  Bread  Soluble  in  the 
Digestive  Juices? 

As  a  preliminary  to  animal  investigation,  it  was  thought  de- 
sirable to  find  out  if  the  aluminium  of  aluminized  bread  was  soluble 
in  artificial  mixtures,  made  to  represent,  as  nearly  as  possible, 
those  juices  with  which  it  would  come  in  contact  during  the 
process  of  digestion. 

To  this  end  a  sample  of  bread  dough  was  made1  and  divided 
into  three  parts.  The  first  of  these  was  baked  very  thoroughly, 
the  second  could  be  called  without  exaggeration  "well  baked," 
while  the  third  was  poorly  baked,  the  interior  being  quite  doughy, 
and  closely  resembling  ordinary  bread.  The  difference  in  degree 
of  baking  is  shown  by  the  appended  summary  of  data  for  the  mois- 
ture content,  determined  on  one-gram  samples  by  drying  to  con- 
stant weight  in  a  vacuum  oven  at  75.  °C. 

Table    1. 

Moisture  content  of  bread  samples.  (%  H2O) 

Very  well  baked.                Well  baked.  Poorly  baked. 

5.84%                               7.02%  16.14% 

5.84%                              7.16%  16.04% 

These  different  grades  of  bread  were  mixed  with  the  extrac- 
tion media  used,  in  the  proportion  of  5  grams  per  liter.  The 
mixtures  were  kept  in  an  incubator  at  body  temperature  for  twen- 
ty-four hours,  and  shaken  ten  times  during  that  period.  The  solid 
material  remaining  was  then  filtered  out,  and  the  aluminium  in  the 
solution  determined  according  to  the  following  procedure : 

Approximately  of  this   composition: 

Flour,  550.  grams. 

Sodium   Chloride,   8.   grams. 

Sugar,   5.   grams. 

Baking  Powder,   16.  grams 

Water,   400.   grams. 

The  baking  powder  used  was  "Parrot  and  Monkey  Brand,"  an  alum 
baking  powder  purchased  in  the  open  market.  It  contained,  according 
to  the  statement  on  the  label,  31.%  anhydrous  soduim  alum.  This  was 
found  to  be  correct. 

3 

425707 


The  solution,  first  partially  evaporated  in  a  porcelain  dish,  was 
taken  to  dryness  in  a  platinum  one.  The  material  was  then  gently 
ignited  in  an  electric  muffle  until  completely  ashed.  This  ash  was 
fused  with  approximately  six  times  its  weight  of  a  mixture  of 
potassium  and  sodium  carbonates,  and  the  melt  dissolved  in  hydro- 
chloric acid  solution.  Repeated  evaporations  with  HC1  were  made 
to  dehydrate  the  silica.  The  residue  was  finally  moistened  with 
cold  concentrated  acid;  after  standing  some  minutes  diluted  with 
water,  warmed,  and  the  silica,  always  small  in  amount,  filtered  off. 
The  aluminium  present  in  the  acid  solution  was  determined  by  the 
procedure  of  Schmidt  and  Hoagland2.  This  method  depends  in 
principle  upon  the  precipitation  of  aluminium  as  phosphate  from 
A  solution  containing  phosphate,  whose  acidity  has  been  reduced 
b>  adding,  first,  ammonium  thiosulphate,  then  ammonium  acetate 
and  acetic  acid.  Reprecipitation  under  similar  conditions  is  neces- 
sary, otherwise  the  precipitate  will  be  contaminated  by  iron  and 
calcium.  The  precipitate  is  first  heated  gently  to  drive  off  the 
accompanying  sulphur,  then  igniated  and  weighed  as  A1PO4. 

This  method  gives  low  results,  but  its  advantages  lie  in  afford- 
ing a  direct  determination  of  aluminium  in  the  presence  of  the 
iron  and  phosphate  always  found  in  biological  material.  The 
method,  as  applied  to  small  amounts  of  aluminium  occurring  in 
biological  substances,  was  found  by  Howe3  and  others  in  this 
laboratory  to  give  satisfactory  results. 

Mellor*  claims  that  water  decomposes  aluminium  phosphate 
slightly  into  a  soluble  aoid  phosphate  and  an  insoluble  basic 
phosphate,  thus  giving  low  results.  This  is  probably  merely  an 
empirical  way  of  describing  a  hydrolytic  process.  By  washing 
the  precipitates  with  dilute  ammonium  phosphate  solution  instead  of 
with  hot  water  as  directed  by  Schmidt  and  Hoagland,  we  found, 
as  one  would  naturally  expect  from  the  common  ion  effect,  that 
less  material  was  lost.  A  final  washing  with  ammonium  nitrate 
solution,  however,  is  advisable,  as  the  presence  of  much  excess  of 
ammonium  phosphate  in  the  precipitate  requires  very  high  and 
prolonged  ignition  to  remove. 

The  bread  samples  were  extracted  in  the  following  four  ways : 

I.  With  distilled  water. 
TI.  With  a  0.2%  solution  of  hydrochloric  acid.     (Represent- 

2Schmidt  and  Hoag-land:  Jour.  Biol.  Chem.  xi,  387,  (1912).  See  also: 
Biochem.  Bull,  v,  (1916). 

3Howe:     Biochem.   Bull,  v,   158,    (1916). 
*Mellor:     Treatise  in  Inorgunic  Analysis,  p.   608,   (1913). 

4 


ing  the  acid  concentration  found  in  the  gastric  juice.) 
III.  With  a  0.2%  solution  of  hydrochloric  acid,  to  which  was 
added  0.1%  of  a  commercial  preparation  of  pepsin.  This 
mixture  is  the  so-called  ''artificial  gastric  juice,"  and  its 
action  will  represent  fairly  completely  the  digestive  pro- 
cesses in  the  stomach. 

IV.  After  allowing  the  bread  to  digest  for  24  hours  in  arti- 
ficial gastric  juice,  sufficient  sodium  carbonate  was  added 
to  furnish  an  excess  of  5  grams  per  liter5,  0.2%  of  a 
commercial  preparation  of  trypsin  was  then  added,  and 
the  mixture  allowed  to  incubate  for  another  24  hours. 

Each  determination  was  made  in  duplicate  with  the  results 
shown  in  the  following  table. 

Table  II. 

Data  showing  the  amounts  of  aluminium  extracted  from  "alu- 
minized  bread"  by  different  solvents,  and  the  relation  of  these 
amounts  to  the  total  quantity  of  aluminium  in  the  bread.  Under 
Column  I.  is  given  a  description  of  the  sample  of  bread  used,  and 
under  Column  II.  is  a  description  of  the  extraction  medium.  The 
wreight  A1PO4  in  milligrams,  representing  the  aluminium  extracted 
from  2.5  grams  of  bread  by  500.  c.c.  of  the  extraction  medium  is 
given  under  Column  III.,  while  Column  IV.  gives  the  mean  of 
the  duplicate  determinations,  expressed  in  terms  of  the  percentage 
of  the  total  aluminium  present,  extracted  by  the  extraction  me- 

Al.   extracted 

dium.      (= x  100.) 

Total  Al 

I.  II.  III.  IV. 

Very  well  Distilled  water  0.2  mg. 

baked  bread.  0.2  2.0% 

0.2%  HC1  9.9 

10.5  86.0% 

0.2%  HC1  +  0.1%  pepsin  11.6 

10.8  93.0% 

0.2%  HC1  +  0.1%  pepsin    1.1 
followed  by  0.5%  Na2CO3 

+  0.2%  trypsin  1.4  11.0% 

0.2%  HC1.  Al  determined    9.1 
in  dialysate,  instead  of 
in  filtrate.  9.8  79.0% 

•^Representing  the  usual  alkalinity  of  the  duodenal   juices. 

5 


0.2%  HCI  +  0.1%  pepsin  10.2 

Al  determined  in 

dialysate.  9.6  83.0% 

Total  aluminium  in  bread  12.0 

sample. 

(2.5   grams)  12.0  (100.0%) 

Well  baked  Distilled  water,  0.9  mg- 

bread  0.8  5.0% 

0.2%  HCI  10.9 

10.5  93.0% 

0,2%  HCI  4-  0.1%  pepsin  11.4 

11.5  100.0% 

0.2%  HCI  +  0.1%  pepsin    0.8' 

followed  by  0.5%  Na2CO3 

+0.2%  trypsin,  0.7  7.0% 

Total  aluminium  in  bread  11.5 

sample. 

(2.5  grams)  11.3  (100.0%) 

Poorly  baked  Distilled  water  1.0  mg- 

bread  0,8  8.0% 

0.2%  HCI.  9.4 

8.8  85.0% 

0.2%  HCI  4-  0.1%  pepsin  10.0 

10.5  96,0% 

0,2%  HCI  4-  0.1%  pepsin    1.0 

followed  by  0.5%  Na2CO3 

4-  0.2%  trypsin.  1.2  10.0% 

.Total  aluminium  in  bread  10.5 

sample. 

(2,5  grams)  10.9  (100.0%) 

Pooly  baked  0.2%  HCI  +  0,1%  pepsin  10.2  mg. 

bread  +  an  10.4  96.0% 

equal  weight  0.2%  HCI  4-0.1%  pepsin  7.4 

of  lean  meat,          Al   determined  in 

dialysate.  7.4  69.0% 

0.2%  HCI  4-  0.1%  pepsin    1.0 

followed  by  0.5%  Na2CO3 

4-  0.2%  trypsin.  0.7  8.0% 

Inspection  of  this  table  will  show  that  but  little  aluminium 
of  the  bread  is  extracted  by  water,  the  solubility  increasing,  how- 
ever, with  decreasing  thoroughness  of  the  baking.  In  the  alkali- 
trypsin  mixture  the  amount  dissolved  is  also  very  small,  although 
larger  than  with  water.  The  same  relationship  also  exists  between 

6 


solubility  and  degree  of  baking.  With  hydrochloric  acid  the  ex- 
traction is  about  two-thirds  of  the  total,  and  the  artificial  gastric 
juice  dissolves  out  practically  all  the  aluminium  present,  the 
amount  dissolved  out  being,  in  both  of  these  cases,  independent  of 
the  degree  of  baking  which  the  material  had  undergone. 

With  the  acidic  extraction  media  the  aluminium  is  dissolved 
in  great  part,  but  not  entirely,  to  form  a  true  solution.  This  can 
be  seen  by  comparing,  in  Table  II,  the  amount  of  aluminium  that 
passes  through'  the  ordinary  filter  paper  used  in  filtering  off  the 
insoluble  bread  residue,  with  that  dialysed  from  a  similar  mixture 
through  a  collodion  membrane.  This  dialysis  increases  the  manip- 
ulative error  and  was  therefore  not  applied  to  the  trypsin-alkali 
and  the  water  extracts,  where  the  quantities  of  available  alumi- 
nium were  much  smaller.  By  adding  to  the  bread,  in  repetitions 
of  the  experiment  described,  an  equal  weight  of  lean  meat,  finely 
divided,  the  presence  of  a  quantity  of  meta-protein,  proposes,  and 
peptones  from  digested  meat  was  found  not  to  affect  the  results 
obtained  from  the  bread  alone,  except  to  increase  the  amount  of 
aluminium  not  in  true  solution  and  therefore  not  dialysable,  but 
still  capable  of  passing  through  a  filter  paper.  This  difference  is 
more  strikingly  indicated  in  subsequent  results  on  aluminium  solu- 
tions containing  large  quantities  of  peptone,  where  fully  half  of 
aluminium  in  "solution"  could  not  be  dialysed  out. 

In  those  cases  where  the  solubility  is  small,  the  percentages 
dissolved  are,  of  course,  useful  only  for  comparison.  The  weights 
of  material  dissolved  probably  represent  the  values,  under  the  con- 
ditions of  the  experiment,  for  a  saturated  solution  of  the  alumi- 
nium compound  in  question  (whatever  that  may  be).  The  total 
amount  dissolved  is  therefore  merely  a  function  of  the  dilution  as 
long  as  any  solid  remains. 

A  series  of  experiments  similar  to  those  above,  were  also  made 
on  a  solution  of-  aluminium  phosphate,  since  the  phosphate  is  the 
form  in  which  previously  ionic  aluminium  would  be  found  after 
entering  the  intestine,  and  it  was  accordingly  decided  to  use  this 
substance  in  feeding  some  of  our  animals. 

One  gram  of  freshly  precipitated,  thoroughly  washed,  moist 
aluminium  phosphate8,  was  dissolved  in  a  liter  of  0.2%  HC1.  On 
the  addition  of  20.  grams  of  Witte  peptone  no  precipitation  oc- 
curred, indicating  that  proteoses  and  peptones  would  not  interfere 
with  the  solubility  of  aluminium  in  the  stomach.  The  aluminium 

6The  material  contained  84.03%  H2O.  An  amount  equal  to  one  gram 
of  the  dried  material  was  employed. 

7 


was  only  partially  in  true  solution,  as  shown  by  the  fact  that  only 
about  half  of  it  was  dialysable.  The  aluminium  was  again  pre- 
cipitated when  an  excess  of  alkali  was  added  equal  to  the  percentage 
of  alkali  found  in  the  intestines.  The  addition  of  the  alkali  as 
above,  but  in  the  presence  of  bile  salts  (0.2%  sodium  taurocholate 
was  used)  was  productive  of  no  difference  in  results.  The  fol- 
lowing table  summarizes  these  findings. 

Table  III. 

In  each  case  the  solution  used  consisted  of  one  gram  of  A1PO, 
dissolved  in  a  liter  of  dilute  HC1,  to  which  the  peptone  was  added 
as  described  above.  Under  Column  I  is  a  description  of  the  sub- 
sequent treatment  of  this  solution,  of  which  50.  c.c.  were  used  in 
the  determinations  under  1.,  and  100.  c.c.  portions  in  the  deter- 
minations under  2.  and  3.  Column  -II.  expresses  the  weight  in 
milligrams  of  A1PO4  in  aliquot  parts  of  the  solutions  after  the  speci- 
fied treatment.  Under  Column  III.  is  given  the  percentage  of  the 

Al.   dissolved 

total  A1PO4,  which  remains  in  solution.    (= x  100.) 

Total  Al. 

I.  II.  III. 

1.  Dialysed  through  24.3  mg. 
collodion. 

Dialysate  used.  24.3  49.0% 

2.  0.5%  Na2CO3  added  in  0.6 
excess  of  amount  necessary  to 

neutralize  acid.     Filtrate  used  0.6  0.6% 

3.  0.2%  Na  taurocholate  -f-  0.9 
excess  of  0.5%  Na2CO3  added. 

Filtrate  used.  0.7  0.8% 

We  conclude,  then,  that  aluminium  is  undoubtedly  rendered 
soluble  and  absorbable,  both  from  aluminized  bread  and  from  alu- 
minium phosphate  by  the  gastric  juice,  and  that  such  soluble 
aluminium  is  not  entirely  rendered  insoluble  by  the  alkaline  intes- 
tinal fluids.  The  locality  of  this  absorptive  process  also  becomes 
evident.  Some  absorption  is  to  be  expected  from  the  stomach 
during  the  digestion  of  aluminized  food,  for  the  gastric  juice  will 
contain  at  that  time,  a  high  concentration  of  aluminium.  Since 
the  stomach  is  not  particularly  well  adapted  to  absorptive  processes 
however,  most  of  the  soluble  aluminium  will  find  its  way  into  the 
intestine,  where  a  great  part  will  again  be  rendered  insoluble. 
This  process,  however,  cannot  be  instantaneous.  The  upper  duo- 

8 


denum  is  known  to  be  frequently  acid,  and  here  absorption  may 
be  rapid,  while  in  the  lower  portions  of  the  intestines  the  amount 
of  metal  taken  up  by  the  body  is  probably  negligible. 

IV. 
Does  Aluminium  Occur  in  the  Tissues  of  Normal  Dogs? 

No  observations  of  the  occurrence  of  aluminium  in  normal 
animal  tissues  have  been  recorded.  It  is  probable  that  while  any 
considerable  quantities,  if  present,  would  have  been  discovered,  at 
the  same  time  traces  might  have  been  as  easily  overlooked,  es- 
pecially when  the  attention  of  investigators  was  not  particularly 
directed  to  this  end. 

Before  determining  whether  aluminium  is  absorbed  by  the  dog 
from  food  containing  it  in  potentially  soluble  form,  it  was  neces- 
sary, therefore,  to  ascertain  whether  aluminium  exists  normally 
in  dog  tissus,  and  if  so,  to  what  extent. 

Accordingly,  dogs  were  selected  which  had  been  fed  for  a 
period  of  about  two  months  on  a  normal  diet.  This  consisted  of 
meat,  cracker  meal,  lard,  and  bone  ash1,  samples  of  which  con- 
stituents, when  extracted  with  artificial  gastric  juice,  according 
to  the  methods  described  in  the  previous  sections,  failed  to  give 
a  filtrate  containing  aluminium. 

From  200.  to  500.  grams  of  blood  were  removed  from  each  dog 
through  a  femoral  artery,  under  local  anaesthesia,  and  the  dogs 
afterwards  maintained  on  the  same  diet.  Three  weeks  later  two 
of  the  dogs  were  bled  to  death,  the  operation  being  conducted  as 
before.  After  exsanguination  was  nearly  complete,  physiological 
salt  solution  was  admitted  to  a  femoral  vein,  and  the  perfusion 
continued  until  practically  all  of  the  blood  had  been  washed  out 
of  the  body  The  washings  were  discarded.  This  perfusion  is 
necessary,  for  were  it  not  done,  and  the  blood  later  found  to  con- 
fain  aluminium,  then  all  the  other  tissues  would  contain  aluminium 
in  proportion  to  their  blood  contents  (which  is  not  accurately  de- 
terminable)  in  addition  to  any  stored  by  the  tissue  itself. 

Various  parts  of  these  two  animals  were  then  selected  and 
prepared  for  analysis.  The  parts  selected  were  those  in  which 
aluminium  might  be  expected,  for  physiological  reasons,  to  occur. 
They  are  also  intended  for  direct  comparison  with  those  dealt  with 

1Hashed  lean  beef  15.  grams  per  kilo  of  body  weight. 
Cracker  meal   2.  grams  per  kilo  of  body  weight. 
I  ard   3.   grams  per  kilo   of  body  weight. 
Bone  ash   1.  gram  per  kilo  of  body  weight. 


ill  section  V,  where  these  selections  will  be  found  to  have  greater 
biological  significance.  Here  we  are  merely  concerned  with  the- 
presence  or  absence  of  aluminium. 

The  tissues  were  freed  from  organic  matter  in  one  of  the  fol- 
lowing ways:  1,  Decomposition  of  the  material  according  to> 
Neumann,  with  nitric  and  sulphuric  acids ;  removal  of  any  nitric- 
acid  by  boiling  with  water,  and  evaporation  of  the  excess  of  sul- 
phuric acid  from  a  silica  or  platinum  dish.  2.  Ignition  in  plat- 
inum, and  subsequent  fusion  of  the  ash  with  sodium  and  potassium 
carbonates. 

The  residues  were  then  treated  precisely  as  those  obtained 
from  the  bread  extracts,  previously  described,  and  the  aluminium 
determined  as  phosphate.  In  the  case  of  bone,  however,  the  fusion 
with  alkali  carbonate  is  not  necessary.  Here  also  the  procedure  of 
Schmidt  and  Hoagland  must  be  modified.  The  large  quantity  of 
calcium  phosphate  present  makes  necessary  a  greater  dilution  of 
the  solution  for  the  first  precipitation.  Comparatively  small 
samples  of  the  material  must  also  be  used,  even  at  the  sacrifice  of 
accuracy.  Working  with  2.5  grams  of  bone  ash,  representing 
about  four  times  that  weight  of  fresh  bone,  the  solution  in  which 
aluminium  phosphate  is  first  precipitated  should  measure  about 
800,  c.c.,  and  the  quantities  of  reagents  be  regulated  accordingly. 
Otherwise  calcium  phosphate  will  contaminate  any  precipitate  of 
aluminium  phosphate.  The  following  results  were  obtained. 

TABLE  IV. 
Data  showing  the  aluminium  content  of  normal  dog  tissues 

Column  I.  designates  the  animal  used;  Column  IL  the  part  of 
the  animal  used;  Column  III.  the  weight  of  the  part  in  grams; 
Column  IV,  the  method  of  decomposition  of  the  tissues;  Column 
V,  the  weight  of  A1PO4  found1  in  milligrams ;  Column  VI.  the 
aluminium  calculated  to  A12O3,  and  Column  VII.  the  aluminium 
calculated  to  milligrams  of  A12O3  per  100,  grams  of  the  original 
material. 


xSix  blank  determinations,  using  the  same  reagents  and  glassware 
were  run  parallel  to  those  recorded  here.  They  gave  the  following 
results  in  milligrams  of  A1PO  ; 

Neumann   Method.  Ignition   Method 

0.2  mg.  0.3  mg. 

0.3  0.3 

0.2  0.3 

The  proper  corrections  have  accordingly  been  subtracted  from  each 
Weight  recorded  in  this  column. 

10 


I. 
c. 

D. 

a 

H. 
E. 


II.          .    III. 

Blood  588. 

513. 

330. 

340. 

"  1st  bleed'g  235. 
"  2d  bleed'g  445. 


IV. 

Neumann 


V.  VI          VIL 

0.1  mg.  0.04mg.  0.01  mg. 


Spleen 
Kidneys  (2) 
Bile  +  Gall 

Bladder 
Muscle  (Leg) 
Claws 

Bone   (Femur) 
Liver 
Blood 

1st  bleeding 

2d  bleeding 
Spleen 
Kidneys  (2) 
Bile+  Gall 

Bladder 
Muscle    (Leg) 
Claws 

Bone   (Femur) 
Ferratin,  from 

liver 
Remainder  of 

liver 


13, 

38. 

15. 
60, 
3.5 
10, 


230. 
320. 

10. 

35. 

6. 
60. 

3. 
10. 

0.130 
200. 


Ignition 


Neumann 


Ignition 


1,1 
0.0 
0,0 
(XI 
0.0 
0,1 


0.0 
0,0 
0.1 
0,0 


0.1 
0.1 
0.0 
0.1 

0.0 
0.0 
0.1 
0.1 


0.46 


0,09 


determination  lost 


determination  lost 


Neumann     0.0 


In  but  one  instance  was  the  amount  of  aluminium  phosphate 
obtained  large  enough  to  exceed  a  reasonable  error  in  making  the 
weighings.  In  this  case  the  blood  undoubtedly  contained  alumi- 
nium, the  presence  of  which  was  confirmed  by  the  qualitative  tests 
described  later.  Unfortunately,  the  animal  died  after  the  first 
bleeding  and  the  body  was  inadvertently  discarded  before  alumi- 
nium was  known  to  be  present  in  the  blood  It  was  therefore  im- 
possible to  continue  work  on  the  other  tissues  of  this  particular 
dog. 

We  regard  a  contamination  of  the  material  during  the  course 
of  analysis,  and  accidental  errors  in  the  manipulation,  as  very  un- 
likely, but,  of  course,  as  possibilities.  The  suggestion  is  also  to  be 
considered  that  the  animal,  whose  previous  history  is  unknown, 
may  have  eaten  aluminized  food  prior  to  the  beginning  of  our  ex- 

11 


periment.  The  fact  that  this  single  result  is  directly  at  variance 
with  all  the  others,  leads  us  to  regard  it  as  exceptional,  to  say  the 
least. 

It  is  reasonable  to  suppose  that  most  of  the  elements  could  be 
found  in  living  matter  if  our  means  for  their  detection  were  deli- 
cate enough.  From  these  results,  however,  we  may  conclude  that 
aluminium  is  present  in  the  normal  dog  in  amounts  that  are  too 
small  usually  for  detection. 

V. 

Is  Aluminium  Absorbed  from  Food  Containing  it  in  Poten- 
tially Soluble  Form? 

Our  work  has  shown  aluminium  to  be  absorbable  from  bread, 
containing  it  supposedly  as  the  hydroxide.  Some  light  on  the  ab- 
sorption of  aluminium  from  the  digestive  tract  was  obtained  in 
section  III.  We  wish,  now,  after  referring  to  the  findings  of 
previous  workers,  to  discuss  the  fate  of  aluminium  after  it  has 
entered  the  body. 

For  this  purpose  a  dog  (G)  was  selected,  whose  blood  content 
of  aluminium  was  previously  found  to  be  too  small  to  be  detected 
by  our  methods.  The  dog  was  then  fed  for  one  month  on  a  diet 
similar  to  the  one  previously  described,  but  with  the  substitution 
of  freshly  precipitated,  moist  aluminium  phosphate  for  the  bone 
ash  of  the  normal  diet.  This  diet  is  somewhat  deficient  in  calcium. 

The  fact  that  aluminium,  even  if  administered  in  a  more  soluble 
form,  would  be  changed  to  phosphate  in  the  upper  small  intestine, 
led  to  the  choice  of  the  phosphate  as  the  form  in  which  to  feed 
the  aluminium.  In  addition,  any  chances  for  acidosis,  or  of  a 
toxic  effect  of  the  anion  attendant  on  feeding  water  soluble  alu- 
minium was  thus  eliminated.  The  body  weight  of  the  dog  at  the 
beginning  of  the  experiment  was  14.0  kilos ;  at  the  end  12.3  kilos. 
A  dose  of  10.  grams  of  aluminium  phosphate  per  day  was  found 
to  be  the  maximum  which  could  be  administered  without  causing 
diarrhea.  The  health  of  the  animal  was  apparently  not  particu- 
larly injured  by  the  experiment,  although  a  decrease  in  body  weight 
and  a  loss  of  appetite  were  apparent,  particularly  toward  the  last. 

The  dog  was  bled  to  death  and  perfused  in  the  manner  already 
explained.  Analyses  of  the  tissues  were  made  for  aluminium  as 
before.  The  results  are  recorded  in  Table  V.  The  presence  of 
aluminium  in  these  precipitates  was  shown  in  each  case  with  Gop- 
pelsroeder's  test.  All  these  precipitates  of  A1PO4  were  white,  in- 

12 


fusible,  and,  when  tested  for  calcium  by  being  moistened  with 
hydrochloric  acid,  and  introduced  on  the  end  of  a  platinum  wire 
into  the  non-luminous  flame  of  a  bunsen  burner,  imparted  no  color 
to  the  flame. 

TABLE  V. 

Amount  of  aluminium  occurring  in  the  tissues  of  dog  G,  fed 
on  freshly  precipitated  A1PO4.  The  different  columns  and  their 
numbering  have  the  same  significance  as  in  Table  IV. 


I.          II. 

III. 

G.     Blood  before 

experiment 

330.  g. 

Blood,  after 

experiment 

(Plasma) 

625. 

Blood,  after 

experiment 

(Corpuscles) 

150. 

Bile  +  Gall 

Bladder 

35. 

Spleen 

30. 

Kidneys    (2) 

85. 

Muscle    (Leg) 

57. 

Bone  (Femur) 

Freed  from 

marrow 

10. 

Bone  marrow 

(Femur) 

8. 

Claws 

4. 

Ferratin,  from 

liver 

0.348 

Remainder  of 

liver 

240. 

IV. 


V. 


VI 


VII. 


Neumann     0.0  mg.  0.00  mg.  0.00  mg. 


1.4 


1.5 

1.5 
0.0 
3.2 
0.2 


0.59 


0.63 

0.63 
0.00 
1.34 
0.08 


0.09 


0.42 

1.80 
0.00 
1.58 
0.15 


Ignition      0.6         0.25         2.5 


1.1 
0.0 

0.5 
Neumann     0.4 


0.46 
0.00 


5.8 
0.0 


0.21       60. 


0.17 


0.07 


As  was  described  in  the  footnote  to  Table  IV.,  blanks  were 
run  and  the  appropriate  corrections  made. 

An  element  in  the  animal  body  can  always  be  detected  when 
present,  by  the  use  of  appropriate  analytical  means.  No  matter 
how  involved  the  chemical  changes  in  which  it  may  take  part,  it 
is  always  possible  to  find  the  element  with  proper  methods.  After 
absorption  such  an  element  would  necessarily  occur  in  the  blood. 
The  blood  analyses  would  therefore  furnish  evidence  for  or  against 
the  passage  of  aluminium  into  the  body.  Since  this  metal  is  for- 
eign to  the  body  there  will  be  a  tendency  toward  elimination,  and 

13 


both  the  excretory  organs  and  their  excretions  may  be  expected 
to  contain  the  metal  in  comparatively  large  amount.  For  this 
reason  the  kidneys,  the  liver,  and  the  contents  of  the  gall  bladder 
were  also  selected  for  analysis..  If,  however,  elimination  is  not 
so  rapid  as  absorption,  storage  must  occur,  and  the  liver,  from 
its  tendency  to  store  metals  would  be  the  most  probable  place  for 
this  accumulation.  On  the  other  hand  there  is  always  the  possi- 
bility that  some  tissue  may  possess  a  particular  retentive  power 
for  the  metal  in  question,  and  to  investigate  this,  samples  were 
taken  of  muscle  (from  the  leg),  of  a  typical  keratin  (the  claws), 
of  bone  (the  femur),  and  of  one  of  the  glands  of  internal  secre- 
tion (the  spleen). 

Our  results  show  most  of  the  aluminium  to  be  speedily  elim- 
inated, through  the  kidneys  and  by  the  liver.  Analyses  of  the  urine 
of  human  beings  fed  with  aluminized  bread,  show  considerable 
aluminium  and  are  in  agreement  with  this  finding1.  There  is, 
however,  a  storage  of  aluminium,  at  least  temporary,  which  is  not 
confined  to  any  one  particular  tissue.  The  distribution  is  not  uni- 
form, however,  and  seems  to  be  roughly  parallel  to  that  of  the 
iron. 

Iron  is  the  only  trivalent  metal  normally  found  in  the  body,  and 
as  far  as  we  know,  it  occurs  principally  combined  with  proteins. 
Would  it  be  possible  for  the  closely  related  trivalent  aluminium  to 
replace  part  of  the  iron  in  such  combinations,  in  much  the  same 
way,  perhaps,  as  this  occurs  in  the  mineral  world?  To  prove  this 
absolutely  would  be  difficult  indeed,  for  tissues  are  like  composite 
rocks  rather  than  single  minerals  with  definite  empirical  formulae, 
and  any  comparison  involving  the  total  amount  of  iron  present 
would  be  utterly  worthless. 

Evidence  pointing  strongly  to  such  replacement,  however,  is 
not  lacking.  If  the  blood  be  mixed,  at  the  time  of  collection,  with 
sodium  oxalate  dissolved  in  physiological  salt  solution,  clotting  is 
prevented,  and  the  corpuscles  may  be  separated  from  the  plasma 
by  contrifuging.  The  concentration  of  aluminium  in  the  corpus- 
cles was  found  to  be  much  higher  than  in  the  iron-free  plasma. 
Now  in  adult  animals  not  suffering  from  anaemia,  the  red  cor- 
puscles are  synthesized  in  the  marrow  of  the  long  bones.  Ac- 
cordingly, the  femur  was  taken  from  the  dog's  body,  split,  and  the 
marrow,  including  as  much  of  the  spongy  portion  of  the  bones  as 
possible,  was  removed  with  a  steel  instrument.  Both  the  bone  and 
the  marrow  contained  aluminium  but  the  latter  did  so  in  ^  ery  large 

TPaper  in  process  of  publication. 

14 


quantities^  The  presence  of  the  metal  in  the  red  corpuscles, 
therefore,  dates  from  the  formation  of  the  cell,  and  can  be  regard- 
ed as  an  original  constituent  rather  than  as  a  subsequently  acquired 
"impurity." 

Furthermore,  the  erythrocytes  are  broken  down  in  the  liver 
and  the  iron  formerly  contained  in  the  haemoglobin  is  stored  there, 
in  a  great  part  as  ferruginous  proteins,  the  so-called  "ferratins.*' 

Ferratin  was  accordingly  prepared  from  the  liver.  The  method 
used  was  that  of  Wohlgemuth2,  which  is  essentially  the  same  as 
the  original  proceedure  of  Schmiedeberg.:! 

The  fresh,  finely  divided  liver  was  covered  with  water,  and 
the  temperature  gradually  increased  to  boiling,  when  the  aqueous 
extract  was  removed.  Subsequent  extractions  with  boiling  water 
were  then  made.  The  protein  was  precipitated  from  the  extract 
with  dilute  acetic  acid  and  purified  by  dissolving  in  ammonia  and 
reprecipitating  with  acid.  The  preparation  was  finally  washed 
with  absolute  alcohol,  with  absolute  ether,  and  dried.  It  was  of  a 
light  yellow  color,  and  contained  on  analysis,  4.3%  Fe2O3,  resem- 
bling the  ferratin  prepared  for  comparison  from  the  liver  of  nor- 
mal dog  F.  (Section  III.),  which,  however,  contained  only  2.4% 
Fe2O3.  The  aluminium  content  of  the  former  preparation,  (from 
dog  G.),  while  not  very  accurately  determined  on  account  of  the 
small  sample  of  material  available,  was  at  any  rate  very  large. 

Aluminium  can  therefore  be  seen  to  follow  the  iron  from  the 
synthesis  of  haemoglobin  in  the  bone  marrow  to  its  disintegration 
in  the  liver  and  the  storage  of  the  iron  as  ferratin.  That  there  is 
an  "aluminium  circulation,"  comparable  to  the  iron  circulation 
well  known  to  physiologists,  is  then  certain.  Were  this  associa- 
tion of  aluminium  with  the  iron  merely  a  physical  one,  it  is  not 
reasonable  to  believe  that  it  would  persist  through  such  profound 
chemical  changes.  The  replacement  of  protein-combined  iron  by 
aluminium,  and  the  formation  therefore  of  "aluminium-proteins/' 
such  as  we  undoubtedly  obtained  in  the  case  of  our  specimen  of 
ferratin,  seems  to  be  the  only  logical  explanation  of  the  facts. 

The  presence  of  aluminium  in  the  bone  itself  is  interesting  as 
recalling  the  observation  of  Papillon,  already  mentioned.  The 
quantity  found  by  him  is  enormous  in  comparison,  however.  It 
will  be  remembered,  that  Papillon's  experiments  were  made  on 
a  young  animal.  The  diet  used  by  us  would  probably  not  affect 

2Wohlgemuth:     Zeit.  Physiol.  Chem  xxxvii,  475,    (1903). 
3Schmiedeberg:     Archiv  fur  Exp.  Path.  u.  Pharm.  xxxiii,  101,  (1894). 

15 


greatly  the  composition  of  the  bones  of  an  adult  animal,  whose 
skeleton  had  already  fully  developed.  It  was  thought  advisable 
therefore,  to  repeat  the  work  on  a  puppy,  where  this  lack  of  bone- 
building  substances  would  be  more  marked,  owing  to  the  absence 
of  milk  from  the  food,  and  where  the  bones  were  still  in  the 
process  of  rapid  growth.  This  experiment  is  still  in  progress 
(May  12,  1917). 

The  principle  of  aiding  substitution  by  creating  a  deficiency  of 
the  material  to  be  replaced  in  the  diet,  was  evidently  not  correctly 
applied  by  Koenig4.  Since  the  aluminium  replaces  the  iron  rather 
than  the  calcium  of  the  body,  a  diet  deficient  in  iron,  not  calcium, 
is  indicated,  and  experiments  on  an  iron-poor  diet  would  make  an 
interesting  chapter  to  the  investigation. 

We  may  conclude  then  that  aluminium  is  absorbed  from  the 
digestive  tract  of  the  dog  in  considerable  quantities  when  fed  as 
aluminium  phosphate. 

Much  of  this  aluminium  is  speedily  eliminated  in  the  bile  and 
the  urine.  Some,  however,  is  retained  in  the  body,  in  all  probabil- 
ity replacing  the  iron  of  ferruginous  proteins,  and  accompanies 
this  iron  in  its  cycle  :  Bone  Marrow — Red  Corpuscles — Ferratin — 
Bone  Marrow.  Still  another  portion  remains  in  the  bones,  and, 
although  to  a  much  smaller  extent  than  found  by  Papillon,  con- 
firms his  results. 

VI. 
Is  Aluminium  Absorbed  by  Dogs  from  Aluminized  Bread? 

The  experiments  described  here  were  undertaken  to  determine 
whether  or  not  aluminium  is  absorbed  in  dogs  fed  on  a  diet  con- 
taining "aluminized  food."  It  was  planned  to  repeat,  in  a  general 
way,  experiments  previously  conducted  in  this  laboratory1,  but  to 
use,  in  the  determination  of  aluminium,  the  method  devised  by 
Schmidt  and  Hoagland2. 

The  experiments  were  performed  on  two  dogs  (A  and  B), 
which  were  fed  aluminized  food  for  a  period  of  about  three 
months.  The  dogs  had  been  previously  maintained  on  a  definite 
diet  of  hashed  lean  beef,  cracker  meal,  infusorial  earth  and  water. 
During  the  experiment  "baking-powder  biscuits,"  made  from  alum 
baking-powder,  according  to  Kahn3,  were  substituted  for  cracker 

*J.  Konig-:  loc.  cit. 
lKahn:     loc.  cit. 

2Gies:     Biochem  Bull,  v,   (1916). 

3Kahn:  loc.  cit.  The  ingredients  used  were  the  same  as  used  in 
preparing  the  bread  used  in  Section  II. 

16 


meal,  and  in  such  quantity  as  to  give  each  dog,  daily,  about  20.  mg. 
of  aluminium  as  A12O3,  per  kilo  of  body  weight. 

Representative  data,  showing  the  general  condition  of  the  dogs 
at  weekly  intervals  throughout  the  experiments,  are  recorded  in 
Table  VI. 

TABLE  VI, 

Data  showing  the  weight  and  condition  of  the  urine  of  dogs 
A  and  B  during  the  experiments.4 

DOG  A  DOG  B 


0  X  G 
&**  G 

Ill 

<D 

Urine 
48    hour   sample 
Vol.   (cc).      Sp.  Gr. 

pi 

Urine 
48    hour   sample 
Vol.   (cc).       Sp.  Grv 

1. 

13.9 

.... 

9.9 

.  .  . 

.... 

2. 

13.fi 

520. 

1.043 

9.3 

425. 

1,023 

3. 

13.4 

550. 

1.044 

8.7 

240, 

1.030 

4. 

13.3 

565. 

1.043 

9.0 

310. 

1.043 

5.5 

13.0 

450. 

1.052 

9.1 

450. 

1,032 

6. 

13.0 

500. 

1.039 

8.9 

390. 

1.035 

7. 

13.1 

460. 

1,036 

8.5 

450.8 

1.037 

8. 

13.0 

590.8 

.... 

8.4 

.  .  . 

.... 

9. 

12.9 

.... 

8.9 

400. 

1.033 

10. 

13.8 

475. 

1,033 

8.8 

580. 

1.027 

11. 

14.0 

650. 

1.032 

8.8 

375. 

1.040 

12. 

14.1 

725. 

1.030 

At  the  end  of  the  feeding  period,  each  dog  was  bled  to  death 
from  a  femoral  artery  and  the  blood  still  remaining  in  the  tissues 
was  washed  out  by  a  perfusion  with  physiological  salt  solution 
directed  into  a  femoral  vein.  The  blood,  also  the  "blood  wash- 
ings," were  analyzed  separately.  Besides  the  blood,  the  liver, 
contents  of  the  urinary  bladder,  and  contents  of  the  gall  bladder, 
of  each  animal,  were  subjected  to  analysis  for  aluminium. 

In  preparation  for  analysis  the  material  was  placed  in  a  Kjel- 
dahl  flask  and  decomposed  by  the  Neumann  process,  with  nitric 
and  sulphuric  acids,  and  was  then  treated  as  outlined  in  III. 

4The  initial  diet  consisted  (per  kilo  of  body  weight)  of  hashed  lean 
beef,  15.  g.,  "baking-powder  biscuits,"  15.  g.,  infusorial  earth,  1.  g., 
water  20.  cc. 

5Lard  added,  1.  g.  per  kilo.      (Dog  A.) 
6Water  increased  to  30.  cc.  per  kilo  per  day. 

17 


The  analytical  results,  obtained  by  the  Schmidt-Hoaglancf 
method,  are  summarized  in  Table  VII. 

TABLE  VII. 

Data  pertaining  to  the  amount  of  aluminium  absorbed,  in  dogs 
fed  on  a  diet  containing  aluminized  biscuit. 

DOG  A. 

Part  Weight  of          Weight7  Weight  of  A12O3 

material  of  Per  100.  g. 

used.  A1PO4  Total.  material 

Blood  960.  g.  1.3  mg.  0.5  mg.  0.06  mg. 

"Blood-washings"  740.8  0.5  0.1 

Urine  (in  bladder)   64.  0.5  0.2  0.35 

Bile  9."  0.2  0.1  0.93 

Liver  320.  0.8  0.3  0.10 

DOG  B. 

Blood  420.  g.  3.0  mg.  1.3mg.  0.30  mg. 

"Blood-washings".  850.  0.8  0.3 

Urine  (in  bladder)    14.  0.6  0.3  1.65 

Bile  3.  0.9  0.4  12.55 

Liver  240.  0.8  0.3  0.14 

The  phosphates  of  aluminium  thus  obtained  were  white  or  at 
most  faintly  tinged  with  yellow.  They  were  infusible  at  white 
heat,  except  occasionally  when  a  colorless  or  slightly  greenish 
glass  was  formed,  suggesting  the  presence  of  traces  of  iron. 
These  precipitates  are  comparatively  insoluble  in  ordinary  re- 
agents, but  hot  concentrated  phosphoric  or  sulfuric  acids  dissolve 
them  readily. 

Three  control  determinations,  by  the  same  method,  were  made 
in  the  glassware  and  with  the  supplies  of  reagents  used  for  the 
estimations  referred  to  in  Table  VII.  The  following  weights  of 
aluminium  phosphate  (mg.)  were  obtained:  0.2,  0.0,  and  0.1;  av- 
erage, 0.1. 

7The  blank  corrections,  referred  to  above,  have  been  applied  to  these 
weights. 

8The  blood  washings  were  mainly  salt  solution,  of  course.  Estimated 
on  the  assumption  that  1-12  of  the  body  weight  was  blood  and  that  all 
was  flushed  out,  the  amount  of  blood  in  the  washings  was  217.  cc.  for 
dog  A  and  310.  cc.  for  dog  B. 

»Bile  and   gall  bladder. 

18 


The  salt  solution  used  in  obtaining  the  "blood  washings,"  and 
the  distilled  water  used  throughout  the  work  were  found  to  be 
free  from  aluminium. 

After  recording  the  weights  of  the  precipitates  of  aluminium 
phosphate  (Table  VII.)  each  precipitate  was  subjected  to  quali- 
tative analysis  for  aluminium.  For  this  purpose  we  used  a  modi- 
fication of  the  Goppelsroeder  test.10 

Goppelsroeder's  test  depends  upon  the  development  of  an  intense 
green  flourescence  when  an  alcoholic  solution  containing  aluminium 
is  mixed  with  "moriii"  dissolved  in  alcohol.  Morin  is  prepared 
from  an  aqueous 'extract  of  fustic  wood.  The  extract  is  evap- 
orated to  small  volume  and  impure  morin  separates  out.  This  is 
.filtered  off,  and  purified  by  fractional  crystallization  from  alcohol. 
Iron  does  not  affect  the  reaction,  if  the  preparation  of  morin  is 
pure,  but  the  test  is  inhibited  by  the  presence  of  some  free  acids, 
although  acetic  acid  in  fairly  large  proportion  or  traces  of  hydro- 
chloric acid  seem,  however,  to  be  without  effect. 

In  order  to  adapt  this  test  to  the  precipitates  of  A1PO4  and, 
at  the  same  time,  to  prevent  access  of  the  small  amounts  of  alumi- 
nium apt  to  be  introduced  during  the  manipulation,  the  fol- 
lowing procedure  was  adopted:  A  few  drops  of  hydrochloric 
acid  solution  were  evaporated  nearly  to  dryness  on  a  watch  glass 
over  a  small  water  bath;  1-2  cc.  of  alcohol  were  added  and  allowed 
to  evaporate  nearly  to  dryness  thus  removing  most  of  the  acid. 
About  1-2  cc.  of  alcohol  were  again  added  and  the  solution  re- 
moved with  a  capillary  pipet  to  a  small  test  tube.  To  this  a  drop 
of  1.%  alcoholic  morin  solution  was  added,  and  the  mixture  al- 
lowed to  stand  for  some  minutes.  When  this  reaction  was  nega- 
tive, the  reagents  and  glassware  failed  to  yield  soluble  aluminium. 
In  the  latter  event,  a  small  amount  of  the  phosphate  precipitate  was 
then  placed  on  the  watch  glass  and  the  whole  process  repeated  in 
the  tested  pipet  and  test  tube.  When  aluminium  was  present  in 
the  alcoholic  solution,  a  strong  green  flourescence  developed  in  a 
few  minutes.  It  was  best  observed  by  reflected  light,  from  a 
source  rich  in  blue  or  violet  rays. 

All  the  precipitates  obtained  were  tested  according  to  this 
technic  and  invariably  contained  aluminium. 

The  ignited  phosphate  was  never  completely  decomposed  by  this 
procedure,  but  suflicinet  A1C13  was  obtained  in  each  test  to  give  a 

10Goppelsroeder:  Zeit.  Anal.  Ch.  vii,  195,  (1868).  See  also  Balls: 
Biochem.  Bull,  v,  (1916). 

19 


distinct  reaction.     This  test  when  conducted  in  this  way,  detects 
0.01  mg.  of  A12O3  in  such  precipitates. 

Unless  all  glassware  used  in  this  test  is  previously  boiled  in 
hydrochloric  acid  solution  and  washed  with  distilled  water,  the 
controls  will  invariably  show  a  content  of  aluminium,  and  the 
small  precipitates  obtained  in  the  "blank  determinations"  may  do 
so  likewise.  In  these  cases,  the  flourescence  will  be  very  much 
less  intense.  Weakly  positive  tests,  therefore,  have  been  uniform- 
ly regarded  as  negative. 

In  reviewing  the  results,  recorded  in  Table  VII.,  and  confirmed 
qualitatively  with  Goppelsroeder's  test,  it  is  interesting  to  note  that 
although  dog  B.  was  the  smaller  animal,  the  quantities  of  alumi- 
nium found  in  his  tissues  were  larger  than  in  the  case  of  dog  A.. 
Both  dogs  were  in  good  health  but  A  was  undoubtedly  the  more 
vigorous  animal. 

Conclusions:  The  blood,  liver,  bile,  and  urine  from  each  of 
two  dogs  fed  upon  a  diet  including  biscuits  baked  with  alum  baking 
powder,  contained  aluminium  in  every  instance. 

These  findings  corroborate  those  of  Steel  and  Kahn.  They 
prove  that  aluminium  is  absorbed  in  dogs  from  a  diet  containing 
aluminized  bread. 

Summary 

We  may  briefly  summarize  our  findings  as  follows : 

1.  The  method  of  Schmidt  and  Hoagland  for  the  determina- 
tion  of  aluminium,   in   which   the   aluminium   is   precipitated  and 
weighed  as  A1PO4,  gives  low  results.     The  addition  of  ammonium 
phosphate  to  the  water  used  for  washing  is  recommended. 

2.  From  bread  baked  with  aluminium  baking  powder  practical- 
ly   all   the   aluminium   is   extracted   by   "artificial   gastric    juice." 
After  gastric  digestion  of  such  bread,   some  aluminium  still   re- 
main dissolved  when  the  duodenal  conditions  affecting  the  digested 
mixture  are  simulated  in  vitro. 

3.  In   normal   dogs,   aluminium   exists,   if   at   all,   in   amounts 
too  small  to  be  demonstrable  by  the  best  available  methods. 

4.  Aluminium  is  absorbed  by  dogs  from  food  containing  alumi- 
nium phosphate,  and  from  bread  baked  with  alum  baking  powder. 
Much  of  this  aluminium  is  speedily  eliminated,  but  some  is   re- 
tained, replacing  part  of  the  iron  occurring  normally  in  the  tissues. 


20 


Acknowledgement 

The  author  desires  to  express  his  very  sincere  thanks  to  Pro- 
fessor William  J.  Gies,  of  Columbia  University,  for  his  kindly 
interest  and  great  assistance  in  this  research, 


Vita 

Arnold  Kent  Balls  was  born  at  Toronto,  Ontario,  Canada, 
April  2,  1891,  graduated  from  the  Central  High  School  of  Phila- 
delphia, Pa.,  in  1908,  received  the  degree  of  B.  S.  in  Chemistry 
from  the  University  of  Pennsylvania  in  1912,  and  studied  under 
the  faculty  of  Pure  Science  in  Columbia  University  1915-17.  The 
author  was  elected  member  of  Sigma  Xi  fraternity  in  1912  and 
member  of  the  Society  of  Experimental  Biology  and  Medicine  in 
1917. 

Previous  Publications: 

The  Electrolytic  Separation  of  Zinc,  Copper  and  Iron  from 
Arsenic.  Jour.  Ind.  and  Eng.  Chem.  viii  p.  26  (1915)  (with  C.  C. 
McDonnell). 

The  accuracy  of  the  Schmidt-Hoagland  method  for  the  deter- 
mination of  Aluminium.  Biochem,  Bull  V,  p.  195  (1916). 


22 


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